Reichelt B Y, Fleet G H
J Bacteriol. 1981 Sep;147(3):1085-94. doi: 10.1128/jb.147.3.1085-1094.1981.
Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 --> 3)-beta-, (1 --> 3)-alpha-, and (1 --> 6)-beta-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 --> 3)-beta-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 --> 3)-beta-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37 degrees C. Glucanase activity was higher in vegetative cells held at 37 degrees C than cells held at 25 degrees C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.
对粟酒裂殖酵母的无细胞提取物、膜组分和细胞壁制剂进行检测，以确定是否存在（1→3）-β-、（1→3）-α-和（1→6）-β-葡聚糖酶活性。在不同生长阶段的细胞中测定了各种葡聚糖酶。仅发现了（1→3）-β-葡聚糖酶活性，且该活性与细胞壁组分相关。对粗酶进行色谱分离，发现了两种内切（1→3）-β-葡聚糖酶，分别命名为葡聚糖酶I和葡聚糖酶II。葡聚糖酶I由分子量分别为78,500和82,000的两个亚基组成，葡聚糖酶II是一条75,000的单一多肽链。尽管两种酶对海带多糖具有相似的底物特异性和相似的水解作用，但葡聚糖酶II对粟酒裂殖酵母的分离细胞壁具有更高的水解活性。基于对细胞壁的不同裂解活性，显示葡聚糖酶II存在于接合细胞中，在孢子形成细胞中含量最高。葡聚糖酶II似乎特别参与接合和孢子形成，因为营养细胞以及非接合和非孢子形成细胞不含这种酶。葡聚糖酶II在接合细胞中的出现可能是由于从头合成酶，因为将营养细胞和接合细胞的提取物混合未显示出激活作用。当接合细胞的壁与孢子形成细胞的壁混合时，葡聚糖酶活性增加。用胰蛋白酶和蛋白水解抑制剂进行的研究表明，葡聚糖酶II在接合细胞中以酶原形式存在。分离出了一种粟酒裂殖酵母的温度敏感突变体，该突变体在37℃时裂解。在37℃下培养的营养细胞中的葡聚糖酶活性高于在25℃下培养的细胞。与野生型菌株不同，该突变体在营养生长期间含有葡聚糖酶II活性，可能是一个调节突变体。
